The invention concerns biallelic markers of the LSR gene, as well as methods and kits for detecting these polynucleotides. The present invention encompasses methods of establishing associations between these markers and obesity or disorders related to obesity. The invention also concerns the genomic sequence and the cDNAs encoding a subunit of LSR and comprising a biallelic marker of the present invention as well as vectors, host cells and transgenic animals comprising said polynucleotides. The invention further concerns modified LSR proteins comprising at least one amino acid change resulting from the biallelic markers of the present invention as well as antibodies which are specific to these modified LSR proteins.
Obesity is a public health problem that is both serious and widespread. One-third of the population in industrialized countries has an excess weight of at least 20% relative to the ideal weight. The phenomenon continues to worsen particularly in regions of the globe where economies are modernizing. In the United States, the number of obese people has escalated from 25% at the end of the 70s to 33% at the beginning of the 90s.
Obesity considerably increases the risk of developing cardiovascular or metabolic diseases. It is estimated that if the entire population had an ideal weight, the risk of coronary insufficiency would decrease by 25% and that of cardiac insufficiency and of cerebral vascular accidents by 35%. Coronary insufficiency, atheromatous disease and cardiac insufficiency are at the forefront of the cardiovascular complications induced by obesity. For an excess weight greater than 30% the incidence of coronary diseases is doubled in subjects under 50 years. Studies carried out for other diseases are equally eloquent. For an excess weight of 20%, the risk of high blood pressure is doubled. For an excess weight of 30%, the risk of developing a non-insulin-dependent diabetes is tripled, and that of hyperlipidemias is multiplied six-fold.
The list of diseases having onsets promoted by obesity includes: hyperuricemia (11.4% in obese subjects, against 3.4% in the general population), digestive pathologies, abnormalities in hepatic functions, and even certain cancers.
Whether the physiological changes in obesity are characterized by an increase in the number of adipose cells, or by an increase in the quantity of triglycerides stored in each adipose cell, or by both, this excess weight results mainly from an imbalance between the quantities of calories consumed and the quantity of calories used by the body. Studies on the causes of this imbalance have been in several directions. Some have focused on studying the mechanism of absorption of foods and therefore the molecules that control food intake and the feeling of satiety. Other studies have characterized the pathways through which the body uses its calories.
The proposed treatments for obesity are of five types. (1) Food restriction is the most frequently used. The obese individuals are advised to change their dietary habits so as to consume fewer calories. Although this type of treatment is effective in the short-term, the recidivation rate is very high. (2) Increased calorie use through physical exercise is also proposed. This treatment is ineffective when applied alone, but it improves weight-loss in subjects on a low-calorie diet. (3) Gastrointestinal surgery, which reduces the absorption of the calories ingested, is effective, but has been virtually abandoned because of the side effects it causes. (4) The medicinal approach uses either the anorexigenic action of molecules involved at the level of the central nervous system, or the effect of molecules that increase energy use by increasing the production of heat. The prototypes of this type of molecule are the thyroid hormones that uncouple oxidative phosphorylations of the mitochondrial respiratory chain. The side effects and the toxicity of this type of treatment make their use dangerous. (5) An approach that aims to reduce the absorption of dietary lipids by sequestering them in the lumen of the digestive tube is also in place. However, it induces physiological imbalances which are difficult to tolerate: deficiency in the absorption of fat-soluble vitamins, flatulence and steatorrhoea. Whatever the envisaged therapeutic approach, the treatments of obesity are all characterized by an extremely high recidivation rate.
The molecular mechanisms responsible for obesity in man are complex and involve genetic and environmental factors. Because of the low efficiency of the current treatments, it is urgent to define the genetic mechanisms which determine obesity, so as to be able to develop better targeted medicaments.
More than 20 genes have been studied as possible candidates, either because they have been implicated in diseases of which obesity is one of the clinical manifestations, or because they are homologues of genes involved in obesity in animal models. Situated in the 7q31 chromosomal region, the OB gene is one of the most widely studied. Its product, leptin, is involved in the mechanisms of satiety. Leptin is a plasma protein of 16 kDa produced by adipocytes under the action of various stimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptin gene; this protein is undetectable in the plasma of these animals. The administration of leptin obtained by genetic engineering to ob/ob mice corrects their relative hyperphagia and allows normalization of their weight. This anorexigenic effect of leptin calls into play a receptor the central nervous system: the ob receptor that belongs to the family of class 1 cytokine receptors. The ob receptor is deficient in obese mice of the db/db strain. The administration of leptin to these mice has no effect on their food intake and does not allow substantial reduction in their weight. The mechanisms by which the ob receptors transmit the signal for satiety are not precisely known. It is possible that neuropeptide Y is involved in this signalling pathway. It is important to specify at this stage that the ob receptors are not the only regulators of appetite. The Melanocortin 4 receptor is also involved since mice made deficient in this receptor are obese (Gura, (1997)).
The discovery of leptin, and the characterization of the leptin receptor at the level of the central nervous system, opened a new route for the search for medicaments against obesity. This model, however, rapidly proved disappointing. With only one exception (Montague et al., (1997)), the genes encoding leptin or its ob receptor, have proved to be normal in obese human subjects. Furthermore and paradoxically, the plasma concentrations of leptin, the satiety hormone, are abnormally high in most obese human subjects.
Clearly there remains a need for novel medicaments that are useful for reducing body weight in humans. Such pharmaceutical compositions advantageously would help to control obesity and thereby alleviate many of the cardiovascular consequences associated with this condition.
The discovery of new genes which are associated to obesity would also allow the design of novel diagnostic and therapeutic tools acting on the lipid metabolism, useful for diagnosing and treating obesity disorders.
The invention is directed, inter alia, to biallelic markers that are located within the LSR genomic sequence. These biallelic markers represent useful tools to identify statistically significant associations between specific alleles of the LSR gene, and obesity, or a disorder related to obesity. Association studies have already shown that a coding mutation in LSR exon 6 influences both fasting and postprandial plasma triglyceride levels in obese adolescent girls. An intronic SNP, located near the splice site determining the LSR subunit, has been associated with insulin and glucose levels. Hence, the biallelic markers of the LSR gene can lead to the identification of new targets for medicaments acting against obesity or obesity-related disorders. Furthermore, they can be used to diagnose a susceptibility to obesity or to identify the cause of obesity for an individual.
The invention also concerns the genomic sequence and the cDNA sequence encoding subunits of LSR, and comprising a biallelic marker of the present invention, as well as vectors, host cells and transgenic animals comprising said polynucleotides. The invention further concerns modified LSR proteins comprising at least one amino acid change resulting from the biallelic markers of the present invention as well as antibodies which are specific to these modified LSR proteins.
In a first aspect, the invention features, an isolated, purified, or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of any one of SEQ ID Nos: 1 to 13, 15, 17, or the complements thereof, wherein said contiguous span comprises an LSR-related biallelic marker. In a preferred embodiment, said LSR-related biallelic marker is selected from the group consisting of Axe2x80x21 to Axe2x80x220, and A1 to A32. In a preferred embodiment, said LSR-related biallelic marker is selected from the group consisting of A2, A15, A16, A17, A21, A23, A24, A26, and A31. Preferably, said LSR-related biallelic marker is selected from the group consisting of A15, A17, and A21.
Alternatively, the invention features an isolated, purified, or recombinant polynucleotide comprising a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos: 1 to 13, 15, 17, or the complements thereof, wherein said contiguous span comprises an LSR-related biallelic marker. In a preferred embodiment said LSR-related biallelic marker is selected from the group consisting of Axe2x80x21 to Axe2x80x220, and A1 to A32. In a preferred embodiment said LSR-related biallelic marker is selected from the group consisting of A2, A15, A16, A17, A21, A23, A24, A26, and A31. Preferably, said LSR-related biallelic marker is selected from the group consisting of A15, A17, and A21.
Alternatively, the invention features any of the above polynucleotides, wherein said contiguous span is 18 to 35 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide. In a preferred embodiment, said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide.
Alternatively, the invention features any of the above polynucleotides, wherein the 3xe2x80x2 end of said contiguous span is present at the 3xe2x80x2 end of said polynucleotide. In a preferred embodiment, the 3xe2x80x2 end of said contiguous span is located at the 3xe2x80x2 end of said polynucleotide and said biallelic marker is present at the 3xe2x80x2 end of said polynucleotide.
Alternatively, the invention features an isolated, purified or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos: 1 to 13, 15, 17, or the complements thereof, wherein the 3xe2x80x2 end of said contiguous span is located at the 3xe2x80x2 end of said polynucleotide, and wherein the 3xe2x80x2 end of said polynucleotide is located within 20 nucleotides upstream of an LSR-related biallelic marker in said sequence. In a preferred embodiment, the 3xe2x80x2 end of said polynucleotide is located 1 nucleotide upstream of said LSR-related biallelic marker in said sequence. Preferably, said polynucleotide consists essentially of a sequence selected from the following sequences: D1 to D32 and E1 to E31.
Alternatively, the invention features an isolated, purified, or recombinant polynucleotide consisting essentially of a sequence selected from the following sequences: B1 to B53, and C1 to C52.
Alternatively, the invention features an isolated, purified, or recombinant polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of any one of SEQ ID Nos: 14, 16, and 18, wherein said contiguous span includes any one of the following: a serine residue at position 363 of SEQ ID NO: 14; an asparagine residue at position 363 of SEQ ID NO: 14; a proline residue at position 420 of SEQ ID NO: 14; an arginine residue at position 420 of SEQ ID NO: 14; an arginine residue at position 519 of SEQ ID NO: 14; a one amino acid deletion at position 519 of SEQ ID NO: 14; a serine residue at position 344 of SEQ ID NO: 16; an asparagine residue at position 344 of SEQ ID NO: 16; a proline residue at position 401 of SEQ ID NO: 16; an arginine residue at position 401 of SEQ ID NO: 16; an arginine residue at position 500 of SEQ ID NO: 16; a one amino acid deletion at position 500 of SEQ ID NO: 16; a serine residue at position 295 of SEQ ID NO: 18; an asparagine residue at position 295 of SEQ ID NO: 18; a proline residue at position 352 of SEQ ID NO: 18; an arginine residue at position 352 of SEQ ID NO: 15 an arginine residue at position 451 of SEQ ID NO: 18; and a one amino acid deletion at position 451 of SEQ ID NO: 18.
Alternatively, the invention features a polynucleotide for use in a hybridization assay for determining the identity of the nucleotide at an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof.
Alternatively, the invention features a polynucleotide for use in a sequencing assay for determining the identity of the nucleotide at an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof.
Alternatively, the invention features a polynucleotide for use in a allele-specific amplification assay for determining the identity of the nucleotide at an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof.
Alternatively, the invention features a polynucleotide for use in amplifying a segment of nucleotides comprising an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof.
Alternatively, the invention features any of the above-described polynucleotides attached to a solid support.
Alternatively, the invention features an array of polynucleotides comprising at least one polynucleotide attached to a solid support. In preferred embodiments the array is addressable.
Alternatively, the invention features any of the above-described polynucleotides further comprising a label.
In a second embodiment, the invention features a recombinant vector comprising any of, or any combination of, the above-described polynucleotides.
In a third embodiment, the invention features a host cell comprising a recombinant vector described above.
In a fourth embodiment, the invention features a non-human host animal or mammal comprising a recombinant vector described above.
In a fifth embodiment, the invention features a method of genotyping comprising determining the identity of a nucleotide at an LSR-related biallelic marker of any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof in a biological sample. In a preferred embodiment, said biological sample is derived from a single subject. In a preferred embodiment, the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said single subject""s genome. In a preferred embodiment, said biological sample is derived from multiple subjects. In a preferred embodiment, the method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining. In a preferred embodiment, said amplifying is performed by PCR. In a preferred embodiment, said determining is performed by a hybridization assay. In a preferred embodiment, said determining is performed by a sequencing assay. In a preferred embodiment, said determining is performed by a microsequencing assay. In a preferred embodiment, said determining is performed by an allele-specific amplification assay.
In a sixth aspect, the invention features a method of estimating the frequency of an allele in a population comprising determining the proportional representation of a nucleotide at an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof in a pooled biological sample derived from said population.
In a seventh aspect, the invention features a method of detecting an association between a genotype and a phenotype, comprising: a) genotyping at least one LSR-related biallelic marker in a trait positive population according to the method described above; b) genotyping said LSR-related biallelic marker in a control population according to the method described above; and c) determining whether a statistically significant association exists between said genotype and said phenotype.
In an eighth aspect, the invention features a method of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising: a) genotyping at least one LSR-related biallelic marker according to a method described above for each individual in said population; b) genotyping a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In preferred embodiments, said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm. In a preferred embodiment, wherein said second biallelic marker is an LSR-related biallelic marker in any one of SEQ ID Nos: 1 to 13, 15, 17 or the complements thereof.
In a ninth aspect, the invention features a method of detecting an association between a haplotype and a phenotype, comprising: a) estimating the frequency of at least one haplotype in a trait-positive population according to the method described above: b) estimating the frequency of said haplotype in a control population according to the method described above; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In a preferred embodiment said control population is a trait negative-population. In a preferred embodiment, said case control population is a random population. In a preferred embodiment, each of said genotyping of a) and b) is performed on a single pooled biological sample derived from each of said populations. In a preferred embodiment said genotyping of a) and b) is performed separately on biological samples derived from each individual in said populations. In a preferred embodiment said phenotype is a disease involving obesity or disorder related to obesity. Preferably said disorder related to obesity is selected from the group consisting of atherosclerosis, insulin resistance, hypertension, hyperlipidemia, hypertriglyceridemia, cardiovascular disease, microangiopathic in obese individuals with Type II diabetes, ocular lesions associated with microangiopathy in obese individuals with Type II diabetes, renal lesions associated with microangiopathy in obese individuals with Type II diabetes, and Syndrome X. In a preferred embodiment of any of the above methods said LSR-related biallelic marker is selected from the group consisting of Axe2x80x21 to Axe2x80x220, and A1 to A32. Preferably said LSR-related biallelic marker is selected from the group consisting of A2, A15, A16, A17, A21, A23, A24, A26, and A31. More preferably said LSR-related biallelic marker is selected from the group consisting of A15, A17, and A21.
In a tenth aspect, the invention features an isolated, purified, or recombinant polypeptide comprising a contiguous span of at least 6 amino acids of any one of SEQ ID Nos: 14, 16, and 18, wherein said contiguous span includes any one of the following: a serine residue at position 363 of SEQ ID NO: 14; an asparagine residue at position 363 of SEQ ID NO: 14; a proline residue at position 420 of SEQ ID NO: 14; an arginine residue at position 420 of SEQ ID NO: 14; an arginine residue at position 519 of SEQ ID NO: 14; a one amino acid deletion at position 519 of SEQ ID NO: 14; a serine residue at position 344 of SEQ ID NO: 16; an asparagine residue at position 344 of SEQ ID NO: 16; a proline residue at position 401 of SEQ ID NO: 16; an arginine residue at position 401 of SEQ ID NO: 16; an arginine residue at position 500 of SEQ ID NO: 16; a one amino acid deletion at position 500 of SEQ ID NO: 16; a serine residue at position 295 of 2xe2x80x2 SEQ ID NO: 18; and asparagine residue at position 295 of SEQ ID NO: 18; a proline residue at position 352 of SEQ ID NO: 18; an arginine residue at position 352 of SEQ ID NO: 18; an arginine residue at position 451 of SEQ ID NO: 18; and a one amino acid deletion at position 451 of SEQ ID NO: 18.
In an eleventh aspect the invention features an isolated or purified antibody composition that selectively binds to an epitope-containing fragment of the above-described polypeptide, wherein said epitope comprises one of the following: a serine residue at position 363 of SEQ ID NO: 14; an asparagine residue at position 363 of SEQ ID NO: 14; a proline residue at position 420 of SEQ ID NO: 14; an arginine residue at position 420 of SEQ ID NO: 14; an arginine residue at position 519 of SEQ ID NO: 14; a one amino acid deletion at position 519 of SEQ ID NO: 14; a serine residue at position 344 of SEQ ID NO: 16; an asparagine residue at position 344 of SEQ ID NO: 16; a proline residue at position 401 of SEQ ID NO: 16; an arginine residue at position 401 of SEQ ID NO: 16; an arginine residue at position 500 of SEQ ID NO: 16; a one amino acid deletion at position 500 of SEQ ID NO: 16; a serine residue at position 295 of SEQ ID NO: 18; an asparagine residue at position 295 of SEQ ID NO: 18; a proline residue at position 352 of SEQ ID NO: 18; an arginine residue at position 352 of SEQ ID NO: 18; an arginine residue at position 451 of SEQ ID NO: 18; and a one amino acid deletion at position 451 of SEQ ID NO: 18.
In an eleventh aspect, the invention features a use of the previously described genotyping methods to determine whether an individual is at risk of developing a detectable trait or whether a detectable trait that an individual suffers from is statistically associated with one or more LSR biallelic markers.
In a twelfth aspect, the invention features a method for determining whether an individual is at risk of developing a detectable trait or whether a detectable trait that an individual suffers from is statistically associated with one or more LSR biallelic markers, comprising obtaining a nucleic acid sample from said individual and determining the identity of the polymorphic base of said one or more LSR biallelic markers in said nucleic acid sample.
In a thirteenth aspect, the invention features a method of using a drug, comprising obtaining a nucleic acid sample from an individual determining the identity of the polymorphic base of one or more LSR biallelic markers in said nucleic acid sample and administering said drug to said individual if said polymorphic base of said one or more LSR biallelic markers is associated with a positive response to treatment with said drug or is not associated with a negative response to treatment with said drug.
In a fourteenth aspect, the invention features a method of screening an individual for inclusion in a clinical trial of a drug comprising: obtaining a nucleic acid sample from said individual; determining the identity of the polymorphic base of one or more LSR balletic markers in said nucleic acid sample; and including said individual in said clinical trial if said polymorphic base of said one or more LSR biallelic markers is associated with a positive response to treatment with said drug or is not associated with a negative response, treatment with said drug.
In a fifteenth aspect, the invention features a method of identifying one or more LSR biallelic markers associated with a detectable trait comprising: determining the frequencies of each allele of said one or more LSR biallelic markers in individuals with said detectable trait and in individuals without said detectable trait; and identifying one or more alleles of said one or more LSR biallelic markers that are statistically associated with said detectable trait.